Study On Ferrous Chelating Modification Of Trichiurus Haumela Offal Hydrolysate By Enzymolysis And Research For Antibacterial Mechanism

In this paper,with the material of offal of Trichiurus haumela,hydrolysate was prepared by using complicated enzyme.Peptides iron(â…¡)chelate was produced with FeCl₂。The effective composition of peptides iron(â…¡)chelate was purified and identified with Sephadex,HPLC and SDS-PAGE.Antibacterial character,antioxidation character and antibacterial mechanism were studied.The studies on material protein peculiarity and its protein fraction showed that the content of Trichiurus haumela offal protein was 16.44%(wet basis),and 78.36%(dry basis).Trichiurus haumela offal was rich in protein,mineral substance and fat,and it has great potential for development. Trichiurus haumela offal consisted of 18 amino acids,in which the content of Glu,Asp,Gly and Ala was up to 57.70%.Trichiurus haumela offal was composed of alkali-soluble protein,stroma protein, myogen,myofibrilar protein and non-protein nitrogen,meanwhile the molecule weight of these protein fraction was myogen＞alkali-soluble protein＞myofibrilar protein＞non-protein nitrogen. Stroma protein had the highest content,which was 75.83%,and the content of other protein were as follows:alkali-soluble protein 16.31%,myofibrilar protein 3.60%,myogen 2.15%.Trichiurus haumela offal was high in total amount of amino acid,but insufficient in essential amino acid, especially Lys,which demonstrated that Trichiurus haumela offal was a poor-quality protein and unfit for making food but seafood-flavor sauce.The optimal conditions of animal protemax,flavorease and comlicated enzymes of enzymes hydrolysis were established by orthogonal trial.The results showed that the optimal conditions were as follows:amount of Trichiurus haumela offal 4.1g/100mL,temperature 40℃,ratio of two enzymes 1:1,amount of enzyme 6×10³IU/l00mL,pH7.0.Compared with single enzyme,complicated enzymes has a notable reduction in dosage,but increase in concentration of substrate.The effect of complicated enzymes was better than single enzyme.The natural pH of mixture solution was close to the optimal pH,so the step of adjusting pH could be left out.The hydrolysis course for the complicated enzymes was described with 2 linear relationship being expressed as follows:y= 5.345ln(x)+2.637,R²=0.990(undefatted);y=0.807x+1.125,R²=0.992(defatted).In order to make bioactive peptide whose DH was usually lower than 20,it spent little time getting the hydrolysate of same DH with the undefatted material compared with defatted material.At the same time,after this process,the fat of Trichiurus haumela offal existed in the form of floating oil,and was easy to remove.So it was unnecessary to be defatted.During the research of preparation of peptide iron(â…¡)chelate,some parameters such as DH,pH, chelating time,chelating temperature,antioxidant category and dosage had both resemblances and differences compared with the other scholars.In this research,the optimum parameters were as follows:DH6%,pH 7.0,Vc 0.1%,chelating time 20min,chelating temperature 20℃.The Fe²⁺had the highest chelating ratio under the conditions mentioned above.The result of ultraviolet scanning showed that there was some new substance-peptides iron(â…¡)chelate.But the qualitative testing showed that this new material was not pure,and it needed to be purified to eliminate peptides that had not integrate with Fe²⁺.Antibacterial testing results showed that the antibacterial effect of peptides iron(â…¡)chelate was subjected to the volume of hydrolysate.It indicated that the peptides iron(â…¡)chelate made from clear solution of 50mL hydrolysate had the best antibacterial effect.Experiment on ultrafiltration showed that hydrolysate with molecular weight more than 5000Da had better antibacterial effect.In this paper,the antibiotic spectrum of peptides iron(â…¡)chelate was studied for the first time with some common food-spoiling microorganisms,the effect of pH value,temperature,metal ion, protease,EC₅₀and the effect when peptides iron(â…¡)chelate was exposed to atmosphere were also investigated.The results showed that the antibacterial activity was better at pH(6.0⁷.0);peptides iron(â…¡)chelate had not good endurance to high temperature;the usual ion such Na⁺and Ca²⁺had different effect,Ca²⁺had greatly effect on peptides iron(â…¡)chelate,but not Na^-.All the proteases could deprive antibacterial effect of peptides iron(â…¡)chelate,especially pepsin.The experiment on EC₅₀indicated that antibacterial effect of peptides iron(â…¡)chelate according to microorganism strain,while peptides iron(â…¡)chelate had good antibacterial effect on Escherichia coli, Ataphylococcus aureus,Bacillus megaterium and Salmonella sp,though effective antibacterial dosage of peptides iron(â…¡)chelate was high,it was reasonable on account of not be purified.After being exposed to atmosphere for several days,peptides iron(â…¡)chelate could not been utilized by microorganism except imbibing because of its deliquescence.It indicated that peptides iron(â…¡) chelate had the ability of degradation-resistance to microorganism excited in atmosphere.Sephadex G-25 and sepahdex G-75 were employed to purify and separate peptides iron(â…¡) chelate and a basic constituent with high antibacterial activity was obtained.This constituent showed a singly active peak in HPLC.SDS-PAGE showed that the molecular weight of puridied peptides iron(â…¡)chelate was about 26kDa.After been purified,MIC of peptides iron(â…¡)chelate to Escherichia coli,Ataphylococcus aureus.Bacillus megaterium and Bacillus subtilis was 0.5%.Half life of peptides iron(â…¡)chelate was 38.18h with the dosage of 500mg by regression analysis.The antibacterial effect was concerned with content of Fe²⁺,and the experiment testified that the higher conten Fe²⁺,the better antibacterial effect,and modification of Fe^2-was critical to antibacterial effect of hydrolysate.Peptides iron(â…¡)chelate had effect of elimination on-OH and H₂O₂,also in oil anti-oxidation.The effect of anti-oxidation was close to Ve.The element construction of purified peptides iron(â…¡)chelate was as follows:C,34.47%;H,5.92%;N,5.50%;O,49.86%;Fe,2.55%. Purified chelate was composed of amino acid and Fe²⁺.IR spectrum showed that purified peptides iron(â…¡)chelate was strongly combined with monodentate covalent bond,and then primary structure of purified peptides iron(â…¡)chelate was put forward.After investigating the effects of purified peptides iron(â…¡)chelate on the growth and cellwall of Escherichia coli and Bacillus subtilis with camera microscope and transmission electron microscopy,the results showed that purified peptides iron(â…¡)chelate could construct channels on the cell wall of G⁺and G^-,then the substance of cell flowed out,cell wall collapsed,and the cell was dead.At the same time,the differences of antibacterial effect among constituents of peptides iron(â…¡) chelate had some connection with their Fe²⁺content,so peptides iron(â…¡)chelate could combine Fe²⁺ competitive with microorganism and play the role of inhibitor.There were two antibacterial mechanisms in purified peptides iron(â…¡)chelate.